The objectives of this project are to develop molecular approaches to investigate pathogen-arthropod interactions of vector-borne diseases of human importance in the United States. The first objective was to develop a sensitive DNA hybridization probe to specifically identify Yersinia pestis, the causative agent of bubonic plague, in experimentally infected fleas. This was achieved in collaboration with K. McDonough and S. Falkow at Stanford University. Primers for use in a polymerase chain reaction (PCR) have been constructed and will be used to detect Y. pestis in experimentally infected fleas. The gene for Fraction 1 has been cloned in Escherichia coli using the lambdaZAPII expression vector. The recombinant E. coli expresses significant amounts of the F1 antigen and protects mice that are challenged with virulent Y. pestis. We will evaluate the feasibility of this in developing a subunit vaccine for plague based on the F1 antigen. The possible role of soft ticks (Argasidae) in maintaining the plague bacillus in nature was examined experimentally using five species of the genus Ornithodoros. Most notably, 80% of Ornithodoros hermsi were infected with Yersinia pestis one month after feeding on infected mice, and viable Y. pestis were detected in 2% of the ticks after one year. Currency, we are completing our work on plague and will expand this project to examine tick-spirochete interactions.